Reactive oxygen species (ROS) produced via mitochondrial respiration are historically considered pro-aging molecules1. By damaging cellular macromolecules such as DNA, ROS promote genome instability and cancer to curtail human health-span and lifespan. However, recent studies have identified many essential signaling functions of ROS, challenging the dogma that these molecules are simply detrimental cellular byproducts. Using the yeast Saccharomyces cerevisiae as a model for post-mitotic cellular aging, we recently demonstrated that elevated levels of mitochondrial ROS (mROS) early in yeast growth elicit an adaptive response that alters mitochondria function to decrease ROS levels late in life and extend chronological lifespan2. Interventions that elevate mROS in C. elegans also extend lifespan3, indicating that adaption to mROS may represent a significant and conserved means to extend lifespan and health-span. The objective of this proposal is to address the mechanism of adaptive mROS signaling in yeast and identify the outcomes of mROS adaption that ultimately determine lifespan. Our preliminary data suggest that mROS activates the DNA damage response, which elicits epigenetic and transcriptional changes to promote genomic stability and extend yeast lifespan. Aim One will identify the mechanism by which mROS activates the DDR by utilizing gene disruption and microscopy to explore candidate DDR factors that may respond to mROS. Additionally, this aim will investigate if mROS activates the DDR via damage to mitochondrial DNA (mtDNA) using assays for mtDNA mutation at early time-points following mROS induction. Aim Two will explore the transcriptional response to mROS mediated by a histone demethylase that we have determined is essential to mROS adaption. We will perform microarray analysis of both an early time-point during mROS adaption and a late time-point following adaption to identify the acute transcriptional changes that prepare yeast for survival in the presence of mROS and the sustained changes that ultimately determine longevity. Finally, Aim Three will investigate the hypothesis that adaption to mROS directly increases mtDNA stability by increasing the activity of mtDNA repair proteins, and that improved mtDNA stability contributes to increased nuclear genome stability and lifespan extension. To investigate this hypothesis, we will utilize gene knockouts and microscopy to determine the requirement and mitochondrial localization of mtDNA repair factors in adaption to mROS. Furthermore, we will determine if the DDR regulates the localization and activity of mtDNA repair proteins. Together, these experiments will increase our understanding of mROS as signaling molecules, and describe the mROS signaling pathway and outcomes of mROS signaling in mechanistic detail. Completion of this proposal will identify genes and processes that respond to mROS to regulate yeast lifespan, and may therefore identify targets for therapies and interventions to increase cellular lifespan and human health-span.